This invention is in the field of plant molecular biology. More specifically, this invention pertains to nucleic acid fragments encoding magnesium chelatase subunit in plants and seeds.
Magnesium chelatase catalyzes the insertion of the magnesium cation (Mg2+) into protoporphyrin IX, the branchpoint in the tetrapyrrole biosynthetic pathways leading to (bacterio)chlorophyll synthesis. In photosynthetic bacteria, magnesium chelatase activity requires three different subunits encoded by the genes bchD, bchH and bchI (Willows, R. D. and Beale, S. I., (1998) J. Biol. Chem. 273:34206-34213). It has been proposed that the BchH subunit initially forms a complex with protoporphyrin IX while the Bch I and BchD subunits form a complex in an ATP-dependent activation step. The BchI-BchD complex then inserts the magnesium cation into the BchH-bound protoporphyrin IX in an ATP-dependent reaction (Willows, R. D. and Beale, S. I. supra).
Similarly in higher plants, three distinct proteins, CHLD, CHLH, and CHLI, encoded by the genes ChlD, ChlH and ChlI respectively (Papenbrock J. et al., (1997) Plant J. 12:981-990) are required for magnesium chelatase activity. They share significant sequence similarity with their bacterial counterparts, further suggesting that the mechanism of magnesium chelation proceeds more or less similarly in plants and bacteria (Guo, R. et al., (1998) Plant Physiol. 116:605-615).
Since magnesium chelatase is an enzyme specific for chlorophyll synthesis, it presents a potential target for discovery and development of herbicides nontoxic to man and animals. Isolation of more genes encoding magnesium chelatase subunits provides a wider array of possible targets, thereby increasing the chances of successfully identifying promising herbicide candidates.
The present invention concerns an isolated polynucleotide comprising a nucleotide sequence selected from the group consisting of: (a) a first nucleotide sequence encoding a polypeptide of at least 50 amino acids having at least 80% identity based on the Clustal method of alignment when compared to a polypeptide selected from the group consisting of SEQ ID NOs:14, 38, 42, and 48; (b) a second nucleotide sequence encoding a polypeptide of at least 100 amino acids having at least 80% identity based on the Clustal method of alignment when compared to a polypeptide selected from the group consisting of SEQ ID NOs:6, 10, and 30; (c) a third nucleotide sequence encoding a polypeptide of at least 100 amino acids having at least 85% identity based on the Clustal method of alignment when compared to a polypeptide of SEQ ID NO:26; (d) a fourth nucleotide sequence encoding a polypeptide of at least 100 amino acids having at least 90% identity based on the Clustal method of alignment when compared to a polypeptide selected from the group consisting of SEQ ID NOs:2, 20, and 34; (e) a fifth nucleotide sequence encoding a polypeptide of at least 100 amino acids having at least 95% identity based on the Clustal method of alignment when compared to a polypeptide of SEQ ID NO:24; (f) a sixth nucleotide sequence encoding a polypeptide of at least 130 amino acids having at least 85% identity based on the Clustal method of alignment when compared to a polypeptide of SEQ ID NO:28; (g) a seventh nucleotide sequence encoding a polypeptide of at least 150 amino acids having at least 80% identity based on the Clustal method of alignment when compared to a polypeptide of SEQ ID NO:32; (h) an eighth nucleotide sequence encoding a polypeptide of at least 250 amino acids having at least 85% identity based on the Clustal method of alignment when compared to a polypeptide of SEQ ID NO:44; (i) a ninth nucleotide sequence encoding a polypeptide of at least 250 amino acids having at least 90% identity based on the Clustal method of alignment when compared to a polypeptide of SEQ ID NO:22; 0) a tenth nucleotide sequence encoding a polypeptide of at least 380 amino acids having at least 90% identity based on the Clustal method of alignment when compared to a polypeptide of SEQ ID NO:8; (k) an eleventh nucleotide sequence encoding a polypeptide of at least 400 amino acids having at least 85% identity based on the Clustal method of alignment when compared to a polypeptide of SEQ ID NO:50; (1) a twelfth nucleotide sequence encoding a polypeptide of at least 400 amino acids having at least 90% identity based on the Clustal method of alignment when compared to a polypeptide of SEQ ID NO:4; (m) a thirteenth nucleotide sequence encoding a polypeptide of at least 750 amino acids having at least 90% identity based on the Clustal method of alignment when compared to a polypeptide of SEQ ID NO:12; (n) a fourteenth nucleotide sequence encoding a polypeptide of at least 1110 amino acids having at least 95% identity based on the Clustal method of alignment when compared to a polypeptide of SEQ ID NO:36; and (o) a fifteenth nucleotide sequence comprising the complement of (a), (b), (c), (d), (e), (f), (g), (h), (i), (0), (k), (1), (m), or (n).
In a second embodiment, it is preferred that the isolated polynucleotide of the claimed invention comprises a nucleotide sequence which comprises a nucleic acid sequence selected from the group consisting of SEQ ID NOs:1, 3, 5, 7, 9, 11, 13, 19, 21, 23, 25, 27, 29, 31, 33, 35, 37, 41, 43, 47, and 49 that codes for the polypeptide selected from the group consisting of SEQ ID NOs:2, 4, 6, 8, 10, 12, 14, 20, 22, 24, 26, 28, 30, 32, 34, 36, 38, 42,44, 48, and 50.
In a third embodiment, this invention concerns an isolated polynucleotide comprising a nucleotide sequence of at least one of 60 (preferably at least one of 40, most preferably at least one of 30) contiguous nucleotides derived from a nucleotide sequence selected from the group consisting of SEQ ID NOs:1, 3, 5, 7, 9, 11, 13, 19, 21, 23, 25, 27, 29, 31, 33, 35, 37, 41, 43, 47, and 49 and the complement of such nucleotide sequences.
In a fourth embodiment, this invention relates to a chimeric gene comprising an isolated polynucleotide of the present invention operably linked to at least one suitable regulatory sequence.
In a fifth embodiment, the present invention concerns a host cell comprising a chimeric gene of the present invention or an isolated polynucleotide of the present invention. The host cell may be eukaryotic, such as a yeast or a plant cell, or prokaryotic, such as a bacterial cell. The present invention also relates to a virus, preferably a baculovirus, comprising an isolated polynucleotide of the present invention or a chimeric gene of the present invention.
In a sixth embodiment, the invention also relates to a process for producing a host cell comprising a chimeric gene of the present invention or an isolated polynucleotide of the present invention, the process comprising either transforming or transfecting a compatible host cell with a chimeric gene or isolated polynucleotide of the present invention.
In a seventh embodiment, the invention concerns a magnesium chelatase subunit polypeptide selected from the group consisting of: (a) a polypeptide of at least 50 amino acids having at least 80% identity based on the Clustal method of alignment when compared to a polypeptide selected from the group consisting of SEQ ID NOs:14, 38, 42, and 48; (b) a polypeptide of at least 100 amino acids having at least 80% identity based on the Clustal method of alignment when compared to a polypeptide selected from the group consisting of SEQ ID NOs:6, 10, and 30; (c) a polypeptide of at least 100 amino acids having at least 85% identity based on the Clustal method of alignment when compared to a polypeptide of SEQ ID NO:26; (d) a polypeptide of at least 100 amino acids having at least 90% identity based on the Clustal method of alignment when compared to a polypeptide selected from the group consisting of SEQ ID NOs:2, 20, and 34; (e) a polypeptide of at least 100 amino acids having at least 95% identity based on the Clustal method of alignment when compared to a polypeptide of SEQ ID NO:24; (f) a polypeptide of at least 130 amino acids having at least 85% identity based on the Clustal method of alignment when compared to a polypeptide of SEQ ID NO:28; (g) a polypeptide of at least 150 amino acids having at least 80% identity based on the Clustal method of alignment when compared to a polypeptide of SEQ ID NO:32; (h) a polypeptide of at least 250 amino acids having at least 85% identity based on the Clustal method of alignment when compared to a polypeptide of SEQ ID NO:44; (i) a polypeptide of at least 250 amino acids having at least 90% identity based on the Clustal method of alignment when compared to a polypeptide of SEQ ID NO:22; (j) a polypeptide of at least 380 amino acids having at least 90% identity based on the Clustal method of alignment when compared to a polypeptide of SEQ ID NO:8; (k) a polypeptide of at least 400 amino acids having at least 85% identity based on the Clustal method of alignment when compared to a polypeptide of SEQ ID NO:50; (1) a polypeptide of at least 400 amino acids having at least 90% identity based on the Clustal method of alignment when compared to a polypeptide of SEQ ID NO:4; (m) a polypeptide of at least 750 amino acids having at least 90% identity based on the Clustal method of alignment when compared to a polypeptide of SEQ ID NO:12; and (n) a polypeptide of at least 1110 amino acids having at least 95% identity based on the Clustal method of alignment when compared to a polypeptide of SEQ ID NO:36.
In an eighth embodiment, the invention relates to a method of selecting an isolated polynucleotide that affects the level of expression of a magnesium chelatase subunit polypeptide or enzyme activity in a host cell, preferably a plant cell, the method comprising the steps of: (a) constructing an isolated polynucleotide of the present invention or a chimeric gene of the present invention; (b) introducing the isolated polynucleotide or the chimeric gene into a host cell; (c) measuring the level of the magnesium chelatase subunit polypeptide or enzyme activity in the host cell containing the isolated polynucleotide; and (d) comparing the level of the magnesium chelatase subunit polypeptide or enzyme activity in the host cell containing the isolated polynucleotide with the level of the magnesium chelatase subunit polypeptide or enzyme activity in the host cell that does not contain the isolated polynucleotide.
In a ninth embodiment, the invention concerns a method of obtaining a nucleic acid fragment encoding a substantial portion of a magnesium chelatase subunit polypeptide, preferably a plant magnesium chelatase subunit polypeptide, comprising the steps of: synthesizing an oligonucleotide primer comprising a nucleotide sequence of at least one of 60 (preferably at least one of 40, most preferably at least one of 30) contiguous nucleotides derived from a nucleotide sequence selected from the group consisting of SEQ ID NOs:1, 3, 5, 7, 9, 11, 13, 19, 21, 23, 25, 27, 29, 31, 33, 35, 37, 41, 43, 47, and 49 and the complement of such nucleotide sequences; and amplifying a nucleic acid fragment (preferably a cDNA inserted in a cloning vector) using the oligonucleotide primer. The amplified nucleic acid fragment preferably will encode a substantial portion of a magnesium chelatase subunit amino acid sequence.
In a tenth embodiment, this invention relates to a method of obtaining a nucleic acid fragment encoding all or a substantial portion of the amino acid sequence encoding a magnesium chelatase subunit polypeptide comprising the steps of: probing a cDNA or genomic library with an isolated polynucleotide of the present invention; identifying a DNA clone that hybridizes with an isolated polynucleotide of the present invention; isolating the identified DNA clone; and sequencing the cDNA or genomic fragment that comprises the isolated DNA clone.
In an eleventh embodiment, this invention concerns a composition, such as a hybridization mixture, comprising an isolated polynucleotide or an isolated polypeptide of the present invention.
In a twelfth embodiment, this invention concerns a method for positive selection of a transformed cell comprising: (a) transforming a host cell with the chimeric gene of the present invention or a construct of the present invention; and (b) growing the transformed host cell, preferably a plant cell, such as a monocot or a dicot, under conditions which allow expression of the magnesium chelatase subunit polynucleotide in an amount sufficient to complement a null mutant to provide a positive selection means.
In a thirteenth embodiment, this invention relates to a method of altering the level of expression of a magnesium chelatase subunit in a host cell comprising: (a) transforming a host cell with a chimeric gene of the present invention; and (b) growing the transformed host cell under conditions that are suitable for expression of the chimeric gene wherein expression of the chimeric gene results in production of altered levels of the magnesium chelatase subunit in the transformed host cell.
A further embodiment of the instant invention is a method for evaluating at least one compound for its ability to inhibit the activity of a magnesium chelatase subunit, the method comprising the steps of: (a) transforming a host cell with a chimeric gene comprising a nucleic acid fragment encoding a magnesium chelatase subunit polypeptide, operably linked to at least one suitable regulatory sequence; (b) growing the transformed host cell under conditions that are suitable for expression of the chimeric gene wherein expression of the chimeric gene results in production of the encoded magnesium chelatase subunit in the transformed host cell; (c) optionally purifying the magnesium chelatase subunit polypeptide expressed by the transformed host cell; (d) treating the magnesium chelatase subunit polypeptide with a compound to be tested; and (e) comparing the activity of the magnesium chelatase subunit polypeptide that has been treated with a test compound to the activity of an untreated magnesium chelatase subunit polypeptide, thereby selecting compounds with potential for inhibitory activity.
Another embodiment of the instant invention is a method for evaluating at least one compound for its ability to inhibit the activity of a magnesium chelatase subunit, the method comprising the steps of: (a) transforming a host cell or plant with a chimeric gene comprising a nucleic acid fragment encoding a magnesium chelatase subunit polypeptide, operably linked to at least one suitable regulatory sequence; (b) growing the transformed host cell or plant under conditions that are suitable for expression of the chimeric gene wherein expression of the chimeric gene results in production of the magnesium chelatase subunit encoded by the operably linked nucleic acid fragment in the transformed host cell or plant; (c) treating the transformed host cell or plant with a compound to be tested; and (d) comparing the viability of the transformed host cell or plant that has been treated with a test compound to the viability of an untreated transformed host cell or plant, thereby selecting compounds with potential for inhibitory activity. Methods for determining viability of cells and plants are well-known to those of ordinary skill in the art.
A further embodiment of the instant invention is a method for conferring, to a host cell or plant, resistance to herbicidal compounds acting on magnesium chelatase, the method comprising the steps of: (a) transforming a host cell or plant with a chimeric gene comprising a nucleic acid fragment encoding a magnesium chelatase subunit polypeptide, operably linked to at least one suitable regulatory sequence; and (b) growing the transformed host cell or plant under conditions that are suitable for expression of the chimeric gene wherein expression of the chimeric gene results in production of the magnesium chelatase subunit encoded by the operably linked nucleic acid fragment in the transformed host cell or plant, and results further in resistance of the transformed host cell or plant to herbicidal compounds acting on magnesium chelatase.